Method of supplying phosphate employing n-phosphorylaminoethyl phosphate

ABSTRACT

N-PHOSPHORYLAMINOETHYL PHOSPHATE IS MADE FROM PHOSPHORIC ACID AND 2-AMINOETHANOL. IT IS USEFUL AS A THERAPEUTIC AGENT FOR PRODUCING HIGH ABSORPTION AND RETENTION OF PHOSPHATE AND CALCIUM WITH EXTREMELY LOW RENAL CALCINOSIS.

United States Patent Oihce 3,749,777 METHOD OF SUPPLYING PHOSPHATE EMPLOY- ING N-PHOSPHORYLAMINOETHYL PHOSPHATE Perttu V. Laakso, Barrington, Ill., assignor to The Kendall Company, Walpole, Mass.

No Drawing. Original application Aug. 15. 1969, Ser. No. 850,636, new Patent No. 3,697,626. Divided and this application Feb. 18, 1972, Ser. No. 227,595

Int. Cl. A61k 27/00 US. Cl. 424-204 1 Claim ABSTRACT OF THE DISCLOSURE N-phosphorylaminoethyl phosphate is made from phosphoric acid and 2-aminoethanol. It is useful as a therapeutic agent for producing high absorption and retention of phosphate and calcium with extremely low renal calcinosis.

This application is a division of copending application Ser. No. 850,636 filed Aug. 15, 1969, now Pat. No. 3,697,- 626, granted Oct. 10, 1972.

This invention relates to N-phosphorylaminoethyl phosphate and its use as a therapeutic agent for introducing phosphate into the bodies of warm-blooded animals.

There have been employed for introducing phosphate into the systems of warm-blooded animals in order to adjust the calcium-phosphorus ratio a variety of phosphate compounds; dosing with these compounds has been employed for treatment of hypercalcemia and osteoporosis, inter alia, and has been described by Tisdall and Harris, Journal American Medical Association, vol. 19, 884-887 (1922); Goldsmith and Ingbar, Journal of Clinical Investigation, vol. 114, 1053 (1965); Cattaneo and Rocchietta, Minerva Medica, vol. 57, 3397-3405 (1966); and Nieper et al. Canadian Pat. No. 760,469.

All of the phosphate theraeputic agents hitherto tried have had various disadvantageous characteristics such as bad taste, or have produced unwanted side effects such as diarrhea, renal calcification, or increase in the sodium or potassium ionic level in the body to an undesirably great extent.

It has now been found that a new compound, N-phos* phorylaminoethyl phosphate is useful as a therapeutic agent for producing both high absorption and high retention of phosphate (and calcium) together with extremely low renal calcinosis as shown by tests on rats. The compound is useful in oral dosage form in amounts from 0.360-0.036 gram daily, per kilogram of body weight approximately 0.072 gram per kg. being preferred and is effective with diets which are low in calcium as well as with those containing the normal amount of calcium. The compound is of relatively low toxicity, the LD in mice being 1.95 g. per kilogram and that in rats being 1.5 g. per kilogram. It may be mixed with food or may be ad ministered in capsule or tablet form. It can be tabletted with conventional tabletting materials such as starch; because of its hygroscopicity it is preferably stored in sealed containers.

The compound N-phosphorylaminoethyl phosphate has the structure:

as verified by elemental analysis, infrared and nuclear magnetic resonance spectroscopy, and sodium hydroxide titer.

The following specific example is intended to illustrate more fully the nature of the present invention without serving as a limitation upon its scope.

3,749,777 Patented July 31, 1973 EXAMPLE 1 There is placed in a three liter resin kettle fitted with a thermometer, dropping funnel, condenser, and magnetic stirrer 2080 g. of phosphoric acid (18.10 moles). There is placed in the dropping funnel 553 g. of 2-aminoethanol (9.05 moles). The aminoethanol is allowed to flow into the kettle very slowly with continuous stirring, the rate of introduction being such that the temperature rises to about 8090 C. and remains within this range. After all of the Z-aminoethanol has been introduced, the kettle is connected to a vacuum pump and all of the free water is distilled off at 80 C. and 0.1 ml. of mercury pressure. At this point the residue in the kettle weighs 2329 g. The temperature of the reaction mixture is then raised gradually to ISO-190 C., and additional water is distilled off until a total of 632 g. has been collected, The residue in the flask weighing 2000 g. is a brown semisolid glassy material. Elemental analysis shows it to have the following composition: carbon 10.87%; hydrogen 4.27%; nitrogen 5.98%; phosphorus 27.85%. The material is stable when stored under anhydrous conditions, but it is hygroscopic and in water it hydrolyzes rapidly to a mixture of aminoethyl phosphate and phosphoric acid, the rate of hydrolysis at C. being approximately 28% per hour at pH 0.8 and approximately 6% per hour at pH 7.2. In acid solution the aminoethyl phosphate which is one of the first stage products of the hydrolysis undergoes further hydrolysis to phosphoric acid and Z-aminoethanol at the rate of approximately 2% per hour at 100 C. at pH 2.7. This further hydrolysis of aminoethyl phosphate does not occur in alkaline solution.

N-phosphorylaminoethyl phosphate is miscible with water in all proportions but is insoluble in non-polar solvents as well as in such common organic solvents as alcohols, acetone, and ether glycols. An aqueous solution at 1% concentration has a pH of 1.9.

The infrared spectrum of the compound contains the following main peaks (in cm. determined by attenuated total reflection method) 2900, 2300, 2200, 2050, 1850, 1690, 1610, 1510, 1460, 1320, 1150, 1060, 860, and 800. The product gives a blue color with ninhydrin reagent. Assay by sodium hydroxide titration gives a purity of 100% (98103%).

The nuclear magnetic resonance spectrum of the compound has the following principal peaks: 2.85, 3.33, 3.75, 4.30, and 5.05 ppm. relative to 60 me. at room temperature using deuterium oxide as solvent.

In order to determine the effectiveness of the new compound as a therapeutic agent, it was mixed in measured proportions with a low phosphorus rat food of the following composition:

Ingredient: Percent by weight Blood fibrin, bovine (washed 6 times) 20.0

Cod liver oil 2.0 Special salt mixture free of phosphorus 7.0 Sucrose 60.0 Hydrogenated vegetable oil 10.0 Vitamin diet fortification mixture 1.0

Analysis of the foregoing diet showed it to contain 0.32% calcium and 0.04% phosphorus by weight. To it was added enough of the new compound to increase the phosphorus content to 0.95%. The diet containing the added compound was fed to a group of six rats for a period of two weeks, during which time the amount of daily food consumption of each rat was measured, and the amount of calcium and phosphorus in the feces and urine of each rat was determined. The absorption was determined by subtracting from the intake the amount excreted in feces. The

retention was calculated by subtracting from the intake the total amount excreted in both feces and urine. Using the above diet in which the percent calcium is 0.32 and the percent phosphorus is 0.95, giving a calciumzphosphorus ratio of 123.0, the percent absorption for calcium and phosphorus respectively is 86.8 and 98.3, and the percent retention is 85.2 and 63.3. After the feeding period, the rats were sacrificed and the calcium and phosphorus content of the kidney was determined both by chemical analysis and histologically by calculating the number of calcium chloranilate crystals in a cross-sectional area near the end of the kidney. Both procedures showed a calcinosis value less than half as great as that for other phosphate esters observed under similar conditions, including aminoethyl phosphate, and also less than half as great as that observed with sodium or potassium phosphate.

in an amount from 0.360 to 0.036 gram per kilogram body weight daily.

References Cited UNITED STATES PATENTS 3,584,125 6/1971 Francis 424204 FOREIGN PATENTS 760,469 6/1967 Canada.

SAM ROSEN, Primary Examiner 

